1. Field of the Invention
The present invention provides methods and compositions for generating novel nucleic acid molecules through targeted spliceosome mediated simple or double trans-splicing. The compositions of the invention include pre-trans-splicing molecules (PTMs, herein also called “TS molecule” for “Trans-Splicing molecules”) designed to interact with a target precursor messenger RNA molecule (target pre-mRNA) and to mediate a simple or double trans-splicing reaction resulting in the generation of a novel chimeric RNA molecule (chimeric RNA). This approach enables to replace whole nucleotide sequences such as exonic sequences in a targeted mRNA and is therefore very interesting to address disorders caused by dominant mutations while preserving levels and tissue specificity. This RNA repair strategy is thus useful to replace mutated nucleic acid sequences into the normal ones and thereby treat many genetic disorders.
In particular, the PTMs of the present invention include those genetically engineered to interact with DMD target pre-mRNA so as to result in correction of DMD genetic defects responsible for the Duchenne muscular dystrophy (DMD).
The compositions of the invention further include recombinant vector systems capable of expressing the PTMs of the invention and cells expressing said PTMs. The methods of the invention encompass contacting the PTMs of the invention with a DMD target pre-mRNA under conditions in which a portion of the PTM is trans-spliced to a portion of the target pre-mRNA to form a mRNA molecule wherein the genetic defect in the DMD gene has been corrected. The methods and compositions of the present invention can be used in gene therapy for correction of neuromuscular disorders such as the Duchenne muscular dystrophy. The principle of this treatment can also be applied to any genetic disease where the pathogenic mutation involves an alteration of the transcript that can be corrected by simple or double trans-splicing.
2. Description of Related Art
DNA sequences in the chromosome are transcribed into pre-mRNAs which contain coding regions (exons) and generally also contain intervening non-coding regions (introns). Introns are removed from pre-mRNAs in a precise process called cis-splicing. Splicing takes place as a coordinated interaction of several small nuclear ribonucleoprotein particles (snRNPs) and many protein factors that assemble to form an enzymatic complex known as the spliceosome (Staley and Guthrie, 1998).
In most cases, the splicing reaction occurs within the same pre-mRNA molecule, which is termed cis-splicing. Splicing between two independently transcribed pre-mRNAs is termed trans-splicing. Trans-splicing was first discovered in trypanosomes and subsequently in nematodes, flatworms and in plant mitochondria, drosophila, mice an humans (Takayuki Horiuchi and Toshiro Aigaki, 2006).
The mechanism of splice leader trans-splicing, which is nearly identical to that of conventional cis-splicing, proceeds via two phosphoryl transfer reactions. The first causes the formation of a 2′-5′ phosphodiester bond producing a ‘Y’ shaped branched intermediate, equivalent to the lariat intermediate in cis-splicing. The second reaction, exon ligation, proceeds as in conventional cis-splicing. In addition, sequences at the 3′ splice site and some of the snRNPs which catalyze the trans-splicing reaction, closely resemble their counterparts involved in cis-splicing.
Trans-splicing may also refer to a different process, where an intron of one pre-mRNA interacts with an intron of a second pre-mRNA, enhancing the recombination of splice sites between two conventional pre-mRNAs. This type of trans-splicing was postulated to account for transcripts encoding a human immunoglobulin variable region sequence linked to the endogenous constant region in a transgenic mouse (Shimizu et al., 1989). In addition, trans-splicing of c-myb pre-RNA has been demonstrated (Vellard, M. et al. 1992) and more recently, RNA transcripts from cloned SV40 trans-spliced to each other were detected in cultured cells and nuclear extracts (Eul et al., 1995). However, naturally occurring trans-splicing of mammalian pre-mRNAs is thought to be a rare event (Finta, C. et al., 2002).
In vitro trans-splicing has been used as a model system to examine the mechanism of splicing by several. Reasonably efficient trans-splicing (30% of cis-spliced analog) was achieved between RNAs capable of base pairing to each other, whereas splicing of RNAs not tethered by base pairing was further diminished by a factor of 10. Other in vitro trans-splicing reactions not requiring obvious RNA-RNA interactions among the substrates were observed for example by Chiara & Reed (1995, Nature). These reactions occur at relatively low frequencies and require specialized elements, such as a downstream 5′ splice site or exonic splicing enhancers.